Acidosis-mediated increase in IFN-γ-induced PD-L1 expression on cancer cells as an immune escape mechanism in solid tumors

Immune checkpoint inhibitors have revolutionized cancer therapy, yet the efficacy of these treatments is often limited by the heterogeneous and hypoxic tumor microenvironment (TME) of solid tumors. In the TME, programmed death-ligand 1 (PD-L1) expression on cancer cells is mainly regulated by Interferon-gamma (IFN-γ), which induces T cell exhaustion and enables tumor immune evasion. In this study, we demonstrate that acidosis, a common characteristic of solid tumors, significantly increases IFN-γ-induced PD-L1 expression on aggressive cancer cells, thus promoting immune escape. Using preclinical models, we found that acidosis enhances the genomic expression and phosphorylation of signal transducer and activator of transcription 1 (STAT1), and the translation of STAT1 mRNA by eukaryotic initiation factor 4F (elF4F), resulting in an increased PD-L1 expression. We observed this effect in murine and human anti-PD-L1-responsive tumor cell lines, but not in anti-PD-L1-nonresponsive tumor cell lines. In vivo studies fully validated our in vitro findings and revealed that neutralizing the acidic extracellular tumor pH by sodium bicarbonate treatment suppresses IFN-γ-induced PD-L1 expression and promotes immune cell infiltration in responsive tumors and thus reduces tumor growth. However, this effect was not observed in anti-PD-L1-nonresponsive tumors. In vivo experiments in tumor-bearing IFN-γ−/− mice validated the dependency on immune cell-derived IFN-γ for acidosis-mediated cancer cell PD-L1 induction and tumor immune escape. Thus, acidosis and IFN-γ-induced elevation of PD-L1 expression on cancer cells represent a previously unknown immune escape mechanism that may serve as a novel biomarker for anti-PD-L1/PD-1 treatment response. These findings have important implications for the development of new strategies to enhance the efficacy of immunotherapy in cancer patients. Supplementary Information The online version contains supplementary material available at 10.1186/s12943-023-01900-0.


Background
Treatment with the programmed cell death-protein 1 (PD-1) immune checkpoint-targeting monoclonal antibodies (mAbs) pembrolizumab or nivolumab has significantly improved the overall survival of patients with various types of cancer.PD-1/PD-L1 blockade represents an approved standard-of-care treatment for patients with metastatic melanoma or non-small cell lung cancer [1,2].Most recently, further PD-1 blocking mAbs have been approved for treatment of patients with advanced squamous cell skin cancer and basal cell carcinomas as well as for patients with advanced or recurrent DNA mismatch repair deficient (dMMR) endometrial cancers [3][4][5].
Despite the impressive improvement in anti-PD-1 therapy for advanced melanoma, only 16% of patients achieve a complete response, and 25% achieve a partial response.Anti-PD-1 treatment results in an estimated 5-year overall survival of 34% in all patients [1], highlighting the importance of studying the mechanisms of treatment response and resistance.
Reliable and robust biomarkers to predict the response to checkpoint inhibitor therapy remain elusive.Key elements that influence the success of checkpoint inhibitor therapies are immune cell infiltration and the tumor mutational burden [6,7].A nonrandomized phase Ib clinical trial with pembrolizumab identified an activated T cell gene expression profile and PD-1 expression as a favorable prognostic biomarker [7].Several studies suggest that patients with tumors overexpressing PD-L1 exhibit a better clinical outcome to anti-PD-L1 therapy [8].PD-L1 expression on cancer cells can vary between different cancer types.Cell-intrinsic, genetic characteristics of the respective cancer cells, such as 9p24.1 amplification leading to increased Janus kinase 2 (JAK2) and PD-L1 expression [9] or oncogenic RAS stabilizing the Pdl1 mRNA [10], explain differences in PD-L1 expression among tumor types.Nevertheless, tumors lacking PD-L1 expression on cancer cells still respond to anti-PD-L1 therapy, indicating that the expression of PD-L1 on cancer cells and tumor-infiltrating immune cells might prevent an antitumor CD8 + T cell response [11][12][13].In addition to cancer cell-intrinsic, genetic PD-L1 regulation, cancer cell-extrinsic factors regulate PD-L1 expression.These factors include IFN-γ secreted by tumor-infiltrating T lymphocytes [14] or hypoxia [15] within the tumor microenvironment (TME).Therefore, cancer cell-intrinsic PD-L1 expression alone might be a poor biomarker to predict the response to PD-1/PD-L1 blockade.Dynamic changes in PD-L1 expression patterns in the TME and infiltrating immune cells may be more relevant for immune escape and predicting the therapy response [13,16].
Resistance to treatment caused by tumor heterogeneity, clonal cooperation [17], and immune inhibition [18] by elements such as acidosis within the TME [19] represent a major challenge in checkpoint inhibitor therapies.Various immune cells, such as T cells, natural killer cells, dendritic cells, and macrophages, show impaired effector functions in an acidic TME, representing a mechanism of tumor immune escape and checkpoint inhibitor resistance [20].Furthermore, lactate produced by cancer cells has been reported to increase PD-L1 expression in human lung cancer cells [21].Knockdown of lactate dehydrogenase-A by a small hairpin RNA increased the efficacy of anti-PD-1 therapy by inducing CD4 + or CD8 + T cell and NK cell recruitment to the tumor [22].A recent study by Kwon et al. suggested that acidosis increases PD-L1 expression via STAT3 activation in MDA-MB-231 cells [23].Further, tumor acidosis suppresses IFN-γ secretion by immune cells representing a separate mechanism of tumor immune escape [24].Hypoxia is often associated with the rapid growth of solid tumors and, triggers the upregulation of hypoxia-inducible factor-1α (HIF-1α).This in turn leads to increased PD-L1 expression in tumor cells.Concurrently, hypoxiainduced acidosis activates multiple signaling pathways, further promoting PD-L1 expression and dampening antitumor immune responses [25][26][27].Necrotic regions within the tumor microenvironment release proinflammatory mediators such as TNF and damage-associated molecular patterns (DAMPs), which in turn stimulate immune cells to upregulate PD-L1 to safeguard against excessive T cell activation [28][29][30].The intricate connection between hypoxia, acidosis, necrosis, and PD-L1 expression fosters immune escape by hindering effector T cell function and promoting the expansion of regulatory T cells [31,32].
The TME is heterogeneous in terms of spatial and temporal dynamic immune cell infiltration and distribution [17].IFN-γ secretion by immune cells, including T cells, B cells, NK cells, and antigen-presenting cells, induces PD-L1 expression by binding to the interferon type II receptor, leading to JAK1, JAK2 and STAT1 activation by phosphorylation [14].IFN-γ-induced PD-L1 expression on the cell membrane is mediated by transcriptional induction and subsequent translation of the Stat1 mRNA by the eukaryotic translation initiation complex eIF4F.Consequently, we hypothesize that immune cell-derived IFN-γ within a tumor region with a neutral tumor pH e affects the dynamics of PD-L1 expression in adjacent acidic tumor regions and thus might represent a novel immune escape mechanism.
To our knowledge, nothing is known so far concerning the effect of IFN-γ in combination with tumor acidosis on PD-L1 expression as an additional prognostic biomarker.
In the present study, we show that acidosis increased IFN-γ-induced PD-L1 expression in cancer cells both in vitro and in vivo.Acidosis-induced PD-L1 expression was mediated by increased STAT1 activity, which relied on the efficient translation of the Stat1 mRNA by elF4F.Furthermore, neutralization of the acidic tumor pH e not only suppressed PD-L1 expression but also increased immune cell recruitment and focal tumor necrosis.Our results highlight a critical role for tumor acidosis in the IFN-γ-mediated induction of PD-L1 expression via enhanced STAT1 phosphorylation and subsequent immune escape of anti-PD-L1 responsive tumors.

In vitro cell culture
For murine MC38 wt and B16-F10 wt cells unbuffered DMEM media was supplemented with 10% FBS, 100 U ml −1 penicillin-streptomycin and NaHCO 3 .An acidic pH e of 6.8 for the continuative in vitro experiments with murine MC38 wt and B16-F10 wt cells at a concentration of 4 mM NaHCO 3 in DMEM cell culture media was chosen.The neutral cell culture media with a concentration of 44.05 mM NaHCO 3 resulted in a pH e of 7.7.Next, for murine CT26 wt and 4T1 wt cells, RPMI-1640 cell culture media supplemented with 10% FBS, 100 U ml −1 penicillin-streptomycin and NaHCO 3 was selected.The cell culture media pH e was again adjusted at 37 °C in a 5% CO 2 atmosphere and determined after 5 h and 24 h.This resulted in a pH e of 6.8 when applying a NaHCO 3 concentration of 1 mM.Respectively a concentration of 23.81 mM (pH e 7.5) or 1 mM NaHCO 3 (pH e 6.8) in RPMI-1640 was applied for all in vitro experiments with murine CT26 wt and 4T1 wt cells.For all in vitro experiments, a consistent terminology was chosen, independent whether cells were maintained in DMEM or RPMI cell culture media.
In one individual experiment (Fig. S2) 5x10 6 MC38 wt tumor cells were cultured at 37 °C in a 5% CO 2 atmosphere in acidic (pH = 6.8), intermediate (pH = 7.4) and neutral (pH = 7.7) media.The pH e of the acidic cell media was adjusted by addition of HCl and the pH e of neutral cell media was adjusted by addition of NaHCO 3 .
Neutral cell culture conditions (pH e 7.7 for DMEM, pH e 7.5 for RPMI) are abbreviated with N, whereas acidic cell culture conditions (pH e 6.8) are abbreviated with A. In the presence of IFN-γ the abbreviations N IFN−γ and A IFN−γ were applied, respectively.

Flow cytometry analysis
For the analysis of cell surface PD-L1 expression, cells were harvested after an incubation with trypsin, washed with PBS (Thermo Fisher Scientific) and passed through a 40 µm cell strainer with a snap cap (Corning, New York, United States).Single-cell suspensions were stained with a Zombie NIR ™ Fixable Viability Kit (Biolegend, San Diego, CA, USA), BV605-CD274 (mouse, clone 10F.9G2, Biolegend) or BV605-CD274 (human, clone 29E.2A3, Biolegend) for 45 min at 4 °C, washed three times with PBS supplemented with 1% FCS and analyzed using a BD LSRFortessa flow cytometer and FlowJo Software (BD Bioscience, San Jose, CA, USA).

Experimental exogenous tumor models
Female C57BL/6 J, C57BL/6N (acidoCEST-MRI measurements) and BALB/c mice were purchased from Charles River Laboratories (Sulzfeld, Germany).B6.129S7-Ifng tm1Ts /J mice [33] were purchased from The Jackson Laboratory and bred in the animal facility in Tübingen.For direct comparison, C57BL/6 J mice bred in the same animal facility were used for the respective experiments.Animals were used at the age of 8 to 10 weeks and maintained in individually ventilated cages with standard rodent pellet food and water or 200 mM NaHCO 3 water (3 days prior to cancer cell inoculation) [24] available ad libitum.Tumors were inoculated by subcutaneous injection (s.c.) of 500,000 MC38 wt , 500,000 MC38 PD−L1−/− , 100,000 CT26 wt or 100,000 CT26 PD− L1−/− cells in PBS into the right flank.B16-F10 wt tumors were inoculated by an intracutaneous (i.c.) injection of 125,000 cells in PBS into the right flank.4T1 wt tumors were inoculated by an injection of 200,000 cells in PBS into the fourth mammary fat pad.Tumor outgrowth was measured in two dimensions, and the tumor volume was calculated using the formula (length*width*width)/2.Tumor-bearing mice were intraperitoneally (i.p.) injected with 200 µg of a PD-L1-blocking antibody (anti-PD-L1, clone 10F.9G2, Bio X Cell, West Lebanon, NH, USA) or IgG2b isotype control (clone LTF-2, Bio X Cell) every third day starting on day 4 (MC38 wt , CT26 wt , B16-F10 wt ) or day 5 (4T1 wt ) after the cancer cell inoculation.Mice were euthanized due to tumor burden (> 15 mm diameter, ulceration) and/or weight loss (> 20%), according to the local guidelines and regulations.

Histology and immunohistochemistry (IHC)
Human metastatic melanoma tissue was fixed with formalin and embedded in paraffin.Tissue samples were cut into 5 μm sections and processed with hematoxylin eosin staining for the histological evaluation.For IHC, sections were cut into 3 µm sections (Leica CM1950).Staining was performed on an immunostainer (BOND-MAX, Leica Biosystems, Wetzlar, Germany) with mAbs against SOX10 (Cell Marque, Rocklin, CA, USA), CD3 (Leica Biosystems, Wetzlar, Germany), Ki67 (Agilent Technologies, Santa Clara, CA, USA) and PD-L1 (Cell Signaling Technology).Negative and positive controls were included.Images were analyzed using a Nikon Eclipse 80i microscope and a Digital Sight DS-U3 camera with NIS-Elements D software.Murine tumors were fixed with 4% formalin and embedded in paraffin.For histology, 3-5 µm-thick sections were cut and stained with hematoxylin and eosin (H&E).H&E stained tumors were scanned with the Ventana DP200 (Roche, Basel, Switzerland) and processed with the Image Viewer MFC Application.Necrosis was evaluated in the H&E tumor scans, and the percentage of necrotic area per total tumor section area is reported (necrosis with an area smaller than 0.01 mm 2 was not considered).Final image preparation was performed with Adobe Photoshop CS6.IHC was performed on selected samples with an automated immunostainer (Ventana Medical Systems, Tucson, AZ, USA) according to the manufacturer's instructions for open procedures with slight modifications.The antibody panel used here included CD3 (Clone SP7; DCS Innovative Diagnostik-Systeme, Hamburg, Germany) and PD-L1 (Cell Signaling Technology, Frankfurt am Main, Germany).Appropriate positive and negative controls were used to confirm the adequacy of the staining.All images were acquired with an Axioskop 2 plus Zeiss microscope equipped with a Jenoptik (Laser Optik System, Jena, Germany) ProgRes C10 plus camera.Image analysis was performed with IMS Client Software.

In vivo acidoCEST MRI measurement
Tumor pH e was determined noninvasively in vivo by chemical exchange saturation transfer (CEST) MRI using iopamidol (Isovue ® , Bracco Diagnostics, Milan, Italy) as an external contrast agent.In vivo MR imaging was performed on a preclinical 7 T BioSpec 70/30 MR scanner with a 1 H volume coil (inner diameter: 86 mm; both Bruker BioSpin, Ettlingen, Germany).Experimental animals were anesthetized with 1.5% isoflurane in air (flow rate: 0.8 l/min).For contrast agent infusion, a catheter was placed into a lateral tail vein.Correct positioning of the animals was achieved with the aid of a short T 1 -weighted FLASH sequence (Table 2).MC38 wt tumors were localized using a standard axial 2D T 2 -weighted TurboRARE protocol (Table 2).Body temperature was monitored with a rectal probe and maintained at 35.0-37.8°C with water-heating systems (Medres, Cologne, Germany).While respiration was monitored and the breathing rate was maintained between 30-50 breaths per minute, respiratory gating was not performed.Aci-doCEST MRI was performed using a previously established CEST FISP acquisition protocol [34].A detailed description of the CEST FISP sequence is provided in Table 2.
The resulting spectra were fit using MATLAB (MAT-LAB R2017b, MathWorks, Inc., Natick, MA, USA) with a previously described Bloch fitting analysis method to generate pH e maps [35].Briefly, the preinjection and postinjection images were averaged at each saturation frequency and smoothed with a Gaussian spatial smoothing algorithm.The resulting averaged and smoothed preinjection image was subtracted from the postinjection image at each saturation frequency to correct for endogenous CEST signals.CEST spectra for each pixel were fitted with the Bloch-McConnell equations for the CEST effects from iopamidol at 4.2 and 5.6 ppm, as well as the water peak; pixels with insufficient contrast (defined as std_noise*2√2/mean_signal) were excluded.Additionally, pixels with pH e values below pH 6.2 and above pH 7.4 were excluded from the analysis.The calculated pH e map was overlaid on the anatomical reference, and data are reported as the average pH e value derived from the sum of individual pixels.

Transcriptome correlation analysis of cutaneous melanoma samples of patients
Transcriptomic data were retrieved via the 'RTCGA' package in R version 3.5.0.The results are based on data generated by TCGA Research Network: https:// www.cancer.gov/ tcga.The 'expressions TCGA' function was used to retrieve the mRNA expression profile of 368 cutaneous melanoma samples.RSEM values were logtransformed and a correlation analysis using the Pearson method was employed for the selected genes.Numbers represent the Pearson coefficients, statistically significant results (p < 0.05) are indicated by the presence of a

Acidosis promotes IFN-γ-induced PD-L1 expression by murine and human cancer cells
IFN-γ derived from activated immune cells (T cells, NK cells and macrophages) located close to acidic tumor regions may promote the expression of the PD-L1 gene in cancer cells in these regions.Consistent with these data, we observed the phenomenon of PD-L1 expression on cancer cells near the T cell-enriched tumor regions in human melanoma metastases (Fig. 1A-H).Histopathological analysis of viable tumor tissue (H&E staining and Ki67 IHC staining) revealed increased PD-L1 expression in SOX10-positive melanoma cells located exclusively near CD3 + T cell-enriched melanoma regions, close to necrotic tumor areas (Fig. 1A-H).Thus, we hypothesized that tumor-infiltrating, IFN-γsecreting immune cells induce PD-L1 expression on cancer cells and thereby mediate immune escape.We used either neutral (N, pH e = 7.5 (RPMI), 7.7 (DMEM)) or acidic (A, pH e = 6.8 (RPMI and DMEM) culture conditions in the presence or absence of IFN-γ and studied PD-L1 expression in cancer cells to investigate the role of acidosis.Acidic conditions were confirmed by determining the mRNA levels of acidosis-associated surrogate markers such as MMP-2 and MMP-9 [36] and enhanced proliferation [37] of MC38 wt cells (Fig. S2A  and B).An acidic pH e was generated by lowering the NaHCO 3 concentration in cell culture media.We first investigated anti-PD-L1-responsive MC38 wt murine colon carcinoma cells to study the effect of basal and induced PD-L1 expression mediated by IFN-γ-secreting immune cells and the acidic TME.IFN-γ treatment under neutral conditions (N IFN−γ ) induced Pdl1 mRNA expression, which was further increased under acidic and IFN-γ conditions (A IFN−γ ).Elevated Pdl1 mRNA expression (Fig. 2A) caused a significant, more than two-fold increase in membrane (Fig. 2B) and total PD-L1 protein levels (Fig. 2C and D), which was also confirmed by fluorescence microscopy (Fig. S3).
Similar to MC38 wt cells, the addition of IFN-γ to acidic culture media increased Pdl1 mRNA (Fig. 2E) and PD-L1 cell surface expression (Fig. 2F) but not total protein expression in murine CT26 wt colon carcinoma cells (Fig. S4B and C) with low basal PD-L1 expression (PD-L1 low , Fig. S5A-C).
Next, we asked whether cancer cells that are nonresponsive to anti-PD-L1 therapy, such as B16-F10 wt melanoma and 4T1 wt mammary carcinoma cells [38], differ in their PD-L1 expression pattern.In sharp contrast to the responsive MC38 wt and CT26 wt cancer cells, no acidosisinduced increase in cell surface PD-L1 expression was observed upon IFN-γ treatment in B16-F10 wt and 4T1 wt cancer cells (Fig. 2G, H).
These results, therefore, indicate that the combination of IFN-γ and acidosis increases the expression of the PD-L1 mRNA, cell surface, and total proteins in various murine and human cancer cells.

Acidosis promotes IFN-γ-induced PD-L1 gene expression by increasing the phosphorylation of STAT1
IFN-γ increased STAT1 protein levels in MC38 wt (Fig. 3A  and B) and CT26 wt cells (Fig. S4B and C) to the same degree under A IFN−γ and N IFN−γ conditions without altering Stat1 mRNA levels (Fig. 3C and S4A).Interestingly, acidosis strongly increased IFN-γ-induced STAT1 phosphorylation in MC38 wt cells (Fig. 3A and B) but not in CT26 wt cells (Fig. S4B).CT26 wt cells revealed only a moderate induction of cell surface PD-L1 expression upon treatment with IFN-γ under acidic conditions (Fig. 2F).Similarly, in human HCA-7 colony 29 cells, the IFN-γ-induced increase in PD-L1 expression under acidic culture conditions (Fig. 2I and S6C) was associated with increased phosphorylation but not the expression of STAT1 (Fig. S6C).Thus, our data indicate that acidosis increases IFN-γ-mediated STAT1 phosphorylation and PD-L1 expression.
We next conducted siRNA-mediated STAT1 knockdown experiments in murine MC38 wt and human HCA-7 colony 29 cancer cells to further elucidate the mechanism of A IFN−γ -induced PD-L1 expression.Knockdown of STAT1 in MC38 wt cells prevented the inducibility of the STAT1 and PD-L1 mRNAs (Fig. 3C and D), as well as membranous (Fig. 3E) and total PD-L1 protein expression (Fig. 3F).In a similar manner knockdown of STAT1 in HCA-7 colony 29 cancer cells prevented from PD-L1 protein synthesis (Fig. S6D).

Acidosis-and IFN-γ-induced PD-L1 expression depends on elF4F
We investigated the role of translational regulation in IFNγ-induced STAT1 and PD-L1 expression under acidic conditions.According to recent studies, silvestrol, an inhibitor of the helicase activity of eukaryotic initiation factor 4A, prevents translation of the Stat1 mRNA [39] (Fig. 3G) and might therefore represent a potential therapeutic option to avoid tumor immune escape via the STAT1-PD-L1 axis.In our experimental setting, acidosis showed no effect on the expression of the elF4F complex components elFA1 and elF4E in MC38 wt cancer cells (Fig. 3H).
Silvestrol upregulated Stat1 mRNA expression under A IFN−γ conditions (Fig. 3I).This is consistent with the results reported by Cerezo et al. that showed enhanced Stat1 mRNA levels after exposure to IFN-γ and silvestrol [40].The increase in Stat1 mRNA levels after stimulation by A IFN−γ and inhibition by silvestrol results from the accumulation of non-translated Stat1 mRNA [41].As shown in Fig. 3D and E, A IFN−γ -induced PD-L1 mRNA and cell surface expression in MC38 wt cells.
Importantly, silvestrol treatment inhibited A IFN−γ -induced PD-L1 mRNA and cell surface upregulation in MC38 wt cells (Fig. 3J and K).The PD-L1 mRNA and cell surface expression were similar to normal pH condition (Fig. 3J and K).Thus, our results support the key role of STAT-1 activation for A IFN−γ -mediated PD-L1 expression.
Following these studies, we asked whether our in vitro findings were also valid in vivo.Thus, the cancer cell lines with different basal, non-stimulated PD-L1 expression patterns studied in vitro (Fig. S5A) were examined in vivo.MC38 wt (PD-L1 high ) and CT26 wt (PD-L1 low ) carcinomas are responsive to anti-PD-L1 mAb therapy, whereas B16-F10 wt (PD-L1 high ) and 4T1 wt (PD-L1 low ) carcinomas show low-/nonresponsive behavior (Fig. S5A) [42], suggesting that the anti-PD-L1 mAb therapy response is independent of basal PD-L1 expression on cancer cells.

NaHCO 3 -treatment neutralizes the extracellular tumor pH e
We used NaHCO 3 to neutralize the tumor pH e (NaHCO 3 ) in vivo to study the effect of the tumor pH e on cancer PD-L1 expression and immune cell recruitment in mice with intact IFN-γ secretion (Control) [24].Noninvasive in vivo acidoCEST MRI measurements confirmed a significant increase in the MC38 wt tumor pH e upon NaHCO 3 treatment (avg.pH e 7.15) compared to MC38 wt tumors from mice receiving regular drinking water (avg.pH e 6.52, control, *P < 0.05; Fig. 4A, B and S7).Furthermore, we identified acidic clusters within MC38 wt tumors, indicating intratumor heterogeneity (Fig. 4A and S7).

Tumor acidosis increases PD-L1 expression in cancer cells
Here we aimed to analyze, whether acidosis-induced PD-L1 expression on cancer cells might represent an immune escape mechanism associated with tumor progression in vivo.MC38 wt tumors from untreated control mice exhibited significantly less necrosis than tumors from the NaHCO 3 solo treatment (Fig. 4C and S8).The NaHCO 3 solo treatment led to an increased percentage of necrotic surface, and this was positively correlated with the rise in pH e , as determined by acidoCEST-MRI (Fig. 4A-C).
In Fig. 4D, H&E staining of an MC38 wt tumor is represented, comparing control-treated and NaHCO 3 -treated experimental mice.Control treatment resulted in a welldefined necrotic area at the center of the MC38 wt tumor (highlighted in pink).In contrast, NaHCO 3 treatment (See figure on next page.)Fig. 3 STAT1 is required for A IFN−γ -induced PD-L1 expression in cancer cells and elF4F inhibition blocks A IFN−γ -mediated PD-L1 expression in cancer cells.A Analysis of pSTAT1, STAT1 and β-actin protein levels in MC38 wt cells treated with acidic media and/or IFN-γ (10 ng ml −1 ) for 12 h and 24 h as determined using Western blotting (B) Densitometry of pSTAT1/STAT1 Western blotting of A. C-F MC38 wt cells were transfected with a control (siCTL) or Stat1-specific siRNA and treated for 24 h with acidic media ± IFN-γ (10 ng ml −1 ) before the assessment of relative (C) Stat1 and (D) Pdl1 mRNA expression normalized to Gapdh, Aldolase and β-actin (n = 3) and (E) cell surface PD-L1 expression (n = 3) using qRT-PCR and flow cytometry, respectively.(A, H) The two signals per condition represent two independent samples (n = 2); two independent experiments showed similar results.Statistics: Tukey's multiple comparison test.F Western blot analyses of pSTAT1, STAT1, PD-L1 and β-actin levels were performed to determine the STAT1 knockdown efficiency (n = 2).Data are presented as the means ± SEM.N = neutral media, A = acidic media, N IFN−γ = neutral media plus IFN-γ, A IFN−γ = acidic media plus IFN-γ.G Schematic representation of the eukaryotic translation initiation complex elF4F composed of elF4G, elF4E and elF4A (inhibited by silvestrol) bound to the 5'UTR of the Stat1 mRNA.H Western blot analysis of elF4A1, elF4E and β-actin levels in MC38 wt cells treated with acidic media and/or IFN-γ (10 ng ml −1 ) for 72 h (n = 2).Relative (I) Stat1 and (J) Pdl1 mRNA expression normalized to Gapdh, Aldolase and β-actin (n = 3) and (K) cell surface PD-L1 expression (n = 3) were determined in MC38 wt cells treated with acidic media and/or IFN-γ (10 ng ml −1 ) in the presence of DMSO (control) or silvestrol (30 nM) for 24 h.Data are presented as the means ± SEM.Statistics: Tukey's multiple comparison test.N = neutral media, A = acidic media, N IFN−γ = neutral media plus IFN-γ, A IFN−γ = acidic media plus IFN-γ induced more extensive and diffuse necrosis within the MC38 wt tumor, which was surrounded by regions of hypoxia, evident from the presence of numerous pyknotic cells (Fig. 4D).The representative PD-L1 IHC image in Fig. 4E reveals that nearly all MC38 wt tumor cells displayed positive staining for PD-L1.Notably, PD-L1 expression was heterogeneous, with some cells showing weak expression while clusters of others exhibited intense PD-L1 expression.The regions with the highest PD-L1 expression were primarily located within the necrotic areas (Fig. 4E).
Within MC38 wt tumors of untreated mice we determined an increased PD-L1 expression on proliferating Ki67 + cells compared to MC38 wt tumors of NaHCO 3 -treated mice (Fig. 4F and G).In contrast, MC38 wt tumors derived from NaHCO 3 -treated mice exhibited increased PD-L1 expression on non-proliferating Ki67 − cells (Fig. 4F and G).
Treatment of MC38 wt tumor-bearing mice which underwent NaHCO 3 and anti-PD-L1 (NaHCO 3 + anti-PD-L1) treatment revealed an increased CD3 + T cell infiltration in the tumor periphery with few scattered CD3 + T cells within the TME (Fig. 4H).Pronounced CD3 + T cell infiltration was also observed in CT26 wt and 4T1 wt tumors from the NaHCO 3 + anti-PD-L1-treated group (Fig. S9).In B16-F10 wt tumors of NaHCO 3 -treated experimental mice, we determined a pronounced infiltrate of tumor-adjacent CD3 + T cells but not in the combined NaHCO 3 -and anti-PD-L1-treatment group (Fig. S9).In 4T1 wt tumors of NaHCO 3 -treated experimental mice we observed a CD3 + T cell infiltrate at the tumor margins of all experimental groups.Nevertheless, the strongest accumulation of CD3 + T cells was found in 4T1 wt tumors of experimental mice upon combined treatment with NaHCO 3 and anti-PD-L1 (Fig. S9).As expected MC38 wt tumors of tumor-bearing IFN-γ −/− mice exhibited no enhancement of CD3 + T cell accumulation as a consequence of NaHCO 3 or anti-PD-L1 treatment (Fig. 4H).
Thus, these results support the hypothesis that acidosis-induced PD-L1 expression on cancer cells represents an unknown mechanism of immune evasion, which anti-PD-L1 antibodies might effectively target.

In vivo consequences of acidosis IFN−γ -mediated PD-L1 expression on cancer cells
In addition, we analyzed the effect of acidosis and IFN-γ induced PD-L1 expression on the in vivo growth of different cancer cell types.MC38 wt and CT26 wt tumors displayed decreased tumor growth in vivo upon NaHCO 3 treatment compared to the control (Fig. 5A and D).In addition, the reduction in tumor volume was more pronounced in CT26 wt tumors with low basal (PD-L1 low ) and only weakly inducible PD-L1 expression (Fig. 5D) than in MC38 wt tumors with high basal (PD-L1 high ) and more pronounced inducible PD-L1 expression (Fig. 5A).As expected, both MC38 wt and CT26 wt tumors responded to anti-PD-L1 treatment.They showed a significant reduction in tumor volume [12].Neither MC38 wt nor CT26 wt tumors Fig. 4 Tumor acidosis increases PD-L1 expression on cancer cells and alleviates the immigration of CD3 + T cells.A Representative pH e maps overlaid on T 2 -weighted axial MRI images of MC38 wt tumor-bearing mice injected with iopamidol (i.v.) at day 10 after the cancer cell injection.Mice with intact IFN-γ signaling received either NaHCO 3 -enriched water three days prior to cancer cell injection or regular drinking water (Control).Individual images of the tumors are shown in Fig. S7.B Average pH e across the whole tumor.The NaHCO 3 treatment significantly increased the tumor pH e measured using acidoCEST MRI (n = 3-4 animals per group; 2 mice were excluded from the quantitative analysis because the measured pH e -values were out of the calibration range, see Fig. S7, statistics: one-tailed Mann-Whitney test).C The percentage of necrosis in MC38 wt tumors from the NaHCO 3 and Control groups was quantified using H&E staining, as shown in Fig. S8.D Representative H&E staining of MC38 wt tumors isolated from experimental mice on day 18 after control or NaHCO 3 treatment.H&E staining show the well-delimited necrotic area in the MC38 wt tumor of a control treated mouse (center, pink area).In contrast, within the MC38 wt tumor of a NaHCO 3 treated mouse the necrosis is diffuse surrounded by areas of hypoxia reflected by abundant pyknotic cells (hyperchromatic; magnitude: 10x; scalebar: 100 µm).E Representative PD-L1 IHC of a MC38 wt tumor isolated from experimental mice on day 18 after control treatment.(magnitude: 12.5x, scalebar 2 mm; insert: 50x; scalebar 500 µm).F Representative fluorescence microscopy images of PD-L1, the proliferation marker Ki67 in MC38 wt tumors isolated on day 18 after treatment with NaHCO 3 and control groups.G PD-L1 expression was quantified in proliferating and non-proliferating cells from MC38 wt  in the NaHCO 3 + anti-PD-L1 treatment group exhibited an additive therapeutic effect (Fig. 5A and D).
B16-F10 wt and 4T1 wt tumors, which were not susceptible to A IFN−γ -mediated PD-L1 induction in vitro (Fig. 2G and  H), did not show a significant reduction in tumor volume in the NaHCO 3 -treated experimental group compared to the untreated control group (Fig. 5F and G).As expected, B16-F10 wt and 4T1 wt tumors showed no or only a slight reduction in tumor volume upon anti-PD-L1 treatment (Fig. 5F and G).
In summary, our results indicate that acidosis and IFNγ-mediated PD-L1 expression on cancer cells represents a previously undescribed novel immune escape mechanism that can be targeted by therapeutic blockade of the PD-L1/ PD-1 axis.Thus, we propose that acidosis and IFN-γinducible PD-L1 expression represents an additional biomarker for the therapeutic response than tumor PD-L1 expression or T cell homing patterns.

Discussion
The present study explored whether extracellular tumor acidosis and IFN-γ-inducible PD-L1 expression represent a mechanism of immune escape and, therefore, a novel biomarker for the therapeutic response.
We show that acidosis further increases IFN-γmediated PD-L1 expression on the surface of various human and murine cancer cell lines.The increase in IFN-γ-mediated PD-L1 expression by acidosis relies on STAT1 gene expression and translation of Stat1 mRNA by the eukaryotic translation initiation factor elF4F, whose subunit, the RNA helicase eIF4A, binds and unwinds a selective subset of mRNAs, including Stat1 [40].Therefore, acidosis enhanced IFN-γ-induced STAT1 activation via phosphorylation, implying a feed-forward mechanism of acidosis in IFN-γ-induced PD-L1 expression and immune escape.
Notably, STAT1 knockdown and the use of the eIF4A subunit inhibitor silvestrol revealed a predominant role of STAT1 translation and phosphorylation in the acidosis-mediated increase in IFN-γ-induced PD-L1 expression.Our findings suggest a hitherto unknown link between acidosis-and IFN-γ-mediated PD-L1 expression that might represent a mechanism of immune escape.The proposed mechanism mediated by elF4A, an essential regulator of STAT1 and therefore PD-L1 expression [40,43,44], upon IFN-γ stimulation and increased STAT1 phosphorylation under acidic conditions opens a window for therapeutic interventions to alleviate immune escape of cancer cells by targeting PD-1/PD-L1 signaling.There is some evidence that PD-L1 expression is mainly regulated by activated STAT1 [45].However, we cannot exclude the possibility that other STAT members including STAT3 may impact membranous PD-L1 expression upon dual stimulation with acidosis and IFNγ.Kwon et al. reported an increased STAT3 phosphorylation under acidic media conditions, which was associated with elevated PD-L1 expression in MDA-MB-231 breast cancer cells.However, it is worth noting that in the referred study co-stimulation with acidic media and IFN-γ has not been investigated.
The relevance of acidification of the TME has been recently reported by Cappellesso et al. which have experimentally proven that inhibition of the bicarbonate transporter SLC4A4 in pancreatic adenocarcinoma cells mitigated acidosis within the TME due to bicarbonate accumulation in the extracellular space.Inhibition of SLC4A4 in combination with immune checkpoint inhibitor treatment was applicable to overcome immunotherapy resistance and to prolong the survival of pancreatic adenocarcinoma bearing mice [48].
Counteracting the acidification of the TME reduced MC38 wt colon adenocarcinoma tumor volumes and increased necrotic regions.Consistent with our findings, Faes et al. reported a significant reduction in the MC38 wt tumor volume and increased necrosis upon NaHCO 3 treatment [49].Interestingly, in lung adenocarcinoma, necrosis significantly correlated with PD-L1 expression [50].Tumor acidosis has been reported to impair IFN-γ secretion by activated cytotoxic CD8 + T cells [24] and NK cells [51].Moreover, acidosis suppresses the release of TNF from macrophages [52] and monocytes [44].Thus, the NaHCO 3 treatment restores the secretion of cytokines [51], leading to increased PD-L1 expression in immune cells [53].Since NaHCO 3 proved intolerable for patients, several other pharmaceutical compounds targeting TME acidification are currently being tested in clinical trials.These compounds target members of the monocarboxylate transporter [54], sodium hydrogen antiporter [55], and carbonic anhydrase families [56].Counteracting the acidification of the TME or selective inhibition of elF4A might open a new window for therapeutic intervention.Notably, since the translation of several oncogenes is also eIF4A-dependent, eiF4A inhibitors, such as silvestrol and rocaglaol, have been proposed as promising anticancer agents [57].
We show that the efficacy of NaHCO 3 and anti-PD-L1 treatment is independent of basal tumor PD-L1 expression and that the induction of PD-L1 expression in response to tumor acidosis or IFN-γ secretion in the TME might represent an additional biomarker for prediction of a potential therapeutic response.Interestingly, CT26 wt tumors with a modest increase in PD-L1 expression upon stimulation with the combination of IFN-γ and acidosis responded better to NaHCO 3 -mediated pH e neutralization than MC38 wt tumors.According to several studies, PD-L1 expression, either on tumor or immune cells, mediates tumor immune escape, rendering total tumor PD-L1 expression more reliable biomarker for predicting the response to anti-PD-L1 therapy [11][12][13]58].Our results suggest that the induction of PD-L1 expression by tumor acidosis and IFN-γ secretion within the TME represents a targetable mechanism of immune escape and determines the response.The two tumor models that did not respond to anti-PD-L1 therapy, B16-F10 wt and 4T1 wt , did not respond to NaHCO 3 -mediated pH e neutralization.Our observation concurs with a study by Pilon-Thomas et al. showing the lack of therapeutic response in NaHCO 3 -treated B16 tumor-bearing mice [24].Our results also strongly correlate with recent literature demonstrating that blocking the PD-1/PD-L1 axis with an anti-PD-1 antibody leads to a substantial reduction in tumor growth of MC38 or CT26 tumor bearing mice [59][60][61].In contrast to our results, Williams et al. have shown that CD8 + T cells efficiently inhibit the growth of IFN-γ receptor 2-or JAK1-deficient B16-F10 tumors which are not sensitive to IFN-γ signaling and subsequent PD-L1 upregulation [17].
Several studies suggest a combined immunohistological assessment of PD-L1 expression and tumor-infiltrating lymphocytes to determine IFN-γ-induced PD-L1 expression in the tumor immune microenvironment [62][63][64].This strategy is promising, as it considers immune cell activity, shaped by tumor acidosis.The TME, the infiltration and activation of T cells [24,65,66] and macrophage polarization [67] are altered by tumor acidosis.In prostate cancer, tumor acidosis drives macrophages into a protumor phenotype [67].Furthermore, a low tumor pH e drives immune cells such as CD8 + T cells into an anergic state characterized by reduced cytolytic activity and cytokine secretion [66].In contrast, tumor pH e neutralization improves the response to anti-CTLA-4 and anti-PD-1 mAb immunotherapies [24].
Our correlation analysis of the transcriptomic data of 368 cutaneous melanoma samples revealed a positive correlation of PD-L1 to IFN-γ, STAT-1, IFN-γ driven CXCL10 and CXCL9 as well as to acidosis driven MMP9.
For validation of our proposed novel PD-L1 biomarker approach in a human cohort, we aim to take tumor biopsies to generate tumor cell suspensions.These tumor cells will be stimulated with IFN-gamma upon acidic and neutral culture conditions and changes in membranous PD-L1 expression analyzed via flow cytometry analysis (before and after stimulation).An additional acidosis related increase in PD-L1 expression upon stimulation with IFN-gamma would predict sensitivity to therapeutic blockade of the PD-1/PD-L1 axis.

Conclusions
In conclusion, our data indicate that combining IFN-γ and acidosis-inducible PD-L1 expression on cancer cells represents a tumor immune escape mechanism.To our knowledge, we are the first to report that the phenomenon and mechanism of joint IFN-γ and acidosis-inducible PD-L1 expression, representing a novel and targetable biomarker for identifying immune therapy responders.
ACT TTC TCC TAT TGT TGA TGG AGC AGC CTT AGT β-actin CGG ATG TCA ACG TCA CAC TT GGC CAG GTC ATC ACT ATT GG Gapdh ACA CAT TGG GGG TAG GAA CA AAC TTT GGC ATT GTG GAA GG Pd-l1 CGC CTG CAG ATA GTT CCC AA ATC GTG ACG TTG CTG CCA TA Stat1 TTG ACG ACC CTA AGC GAA CT TCA AAT TCG GGG CCC ACT AT Mmp-2 CAC ACC AGG TGA AGG ATG TG AGG GCT GCA TTG CAA ATA TC Mmp-9 CGT CGT GAT CCC CAC TTA CT AAC ACA CAG GGT TTG CCT TC human Aldolase AAT GTT CTG GCC CGT TAT GC CCA GGT AGA TGT GGT GGT CA β-actin ACT CTT CCA GCC TTC CTT CC TCT CCT TCT GCA TCC TGT CG Gapdh CCA GAA CAT CAT CCC TGC CT CCT GCT TCA CCA CCT TCT TG Pd-l1 GTG CCG ACT ACA AGC GAA TT CTT GGA ATT GGT GGT GGT GG Stat1 GTG GTA CGA ACT TCA GCA GC CAT GAA AAC GGA TGG TGG CA

Fig. 1 Fig. 2 A
Fig. 1 PD-L1 is expressed on melanoma cells located close to T cell-enriched tumor regions.H&E-stained human metastatic melanoma tissues (A, B) with necrotic tissue regions (black asterisk + encircled with white dashed lines).B-F Shows the magnification of the identical region of the tumor as indicated by the black rectangle (dashed lines) in (A).B-F In the viable melanoma tumor tissue (black asterisk), C SOX-10-expressing melanoma cells were discriminated from the (D) CD3 + T cell infiltrate.In addition, viable tumor regions were discriminated from necrotic tumor regions based on (E) Ki67 expression patterns.IHC showed that tumor regions with pronounced (F) PD-L1 expression (black rectangle) were located near the tumor immune cell infiltrate that was identified based on the cell morphology.G At higher magnification (white rectangle) it is visible that PD-L1 positive cells also express SOX-10.The immunofluorescence double staining of PD-L1 and SOX10 (H) of the serial section (F) shows the same region of the tumor (black rectangle) with PD-L1 positive cells surrounding an immune cell infiltrate.Scale bars: 500 μm (A), 100 μm (B-F), 50 μm (H), 20 μm (G)

Fig. 5
Fig.4 Tumor acidosis increases PD-L1 expression on cancer cells and alleviates the immigration of CD3 + T cells.A Representative pH e maps overlaid on T 2 -weighted axial MRI images of MC38 wt tumor-bearing mice injected with iopamidol (i.v.) at day 10 after the cancer cell injection.Mice with intact IFN-γ signaling received either NaHCO 3 -enriched water three days prior to cancer cell injection or regular drinking water (Control).Individual images of the tumors are shown in Fig.S7.B Average pH e across the whole tumor.The NaHCO 3 treatment significantly increased the tumor pH e measured using acidoCEST MRI (n = 3-4 animals per group; 2 mice were excluded from the quantitative analysis because the measured pH e -values were out of the calibration range, see Fig.S7, statistics: one-tailed Mann-Whitney test).C The percentage of necrosis in MC38 wt tumors from the NaHCO 3 and Control groups was quantified using H&E staining, as shown in Fig.S8.D Representative H&E staining of MC38 wt tumors isolated from experimental mice on day 18 after control or NaHCO 3 treatment.H&E staining show the well-delimited necrotic area in the MC38 wt tumor of a control treated mouse (center, pink area).In contrast, within the MC38 wt tumor of a NaHCO 3 treated mouse the necrosis is diffuse surrounded by areas of hypoxia reflected by abundant pyknotic cells (hyperchromatic; magnitude: 10x; scalebar: 100 µm).E Representative PD-L1 IHC of a MC38 wt tumor isolated from experimental mice on day 18 after control treatment.(magnitude: 12.5x, scalebar 2 mm; insert: 50x; scalebar 500 µm).F Representative fluorescence microscopy images of PD-L1, the proliferation marker Ki67 in MC38 wt tumors isolated on day 18 after treatment with NaHCO 3 and control groups.G PD-L1 expression was quantified in proliferating and non-proliferating cells from MC38 wt tumor regions.Statistics: two-tailed Mann-Whitney test.H Representative CD3 immunohistochemistry of s.c.MC38 wt tumors derived from wild-type C57BL/6 J (day 18) or IFN-γ −/− mice (day 17) after the four different treatment conditions.Animals received regular drinking water (control), NaHCO 3 in water (NaHCO 3 ), anti-PD-L1 mAb or NaHCO 3 & anti-PD-L1 mAb.N = 3 -4 representative tumors of each experimental group were subjected to CD3 staining.(magnitude: 400x; scalebar: 100 µm) (See figure on next page.)

Table 2
Acquisition parameters for MR imaging Whitney test was applied.Statistically significant differences in the tumor pH e determined using acidoCEST MRI between the NaHCO 3 -treated neutral IFN−γ and untreated acidosis IFN−γ group were determined using a one-tailed Mann-Whitney test, as only the increase in tumor pH e upon NaHCO 3 treatment was tested for statistical significance.P-values < 0.05 were considered significant (*).